Background
Metastatic spread of tumor cells is the most lethal aspect of cancer and often occurs
via the lymphatic vessels, whereas lymphangiogenesis refers to the formation of lymphatic vessels from preexisting lymphatic vessels, which plays an important role in promoting growth and metastatic spread of tumor cells [
25]. The tumor-associated lymphatic vessel, also referred to as tumor lymphangiogenesis, is the growth of newly formed lymphatic vessels in cancer; this process with multiple steps is similar to the well-known mechanism of angiogenesis including endothelial cell proliferation, migration, rearrangement and tube formation, along with degradation, reconstruction and production of extracellular matrix; thus tumor lymphangiogenesis acts as a conduit by which disseminating tumor cells access regional lymph nodes and form metastases [
25,
31,
43]. VEGF family, which consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PGF), contributes to vasculogenesis composed of neoangiogenesis and lymphangiogenesis. Roskoski R Jr. reviewed the interaction of several ligands and VEGF family of receptors, which consists of three protein-tyrosine kinases (VEGFR-1,-2 and-3) and two non-protein kinase co-receptors (neuropilin-1,-2) [
39]. Extensive studies have showed that tumor- or stromal-secreted cytokines such as VEGF-C and VEGF-D, and their cognate receptor tyrosine kinase VEGFR-3 located on LECs are critical regulators of lymphangiogenesis, these molecules advance or regulate proliferation, migration, metastasis and survival of LECs, growth of new lymphatic capillaries and lymphatic tube formation in tumorigenesis, thus promote metastatic spread of tumor cells to lymph nodes [
20,
28]. Therefore, inhibition of tumor lymphangiogenesis or its VEGF-C,-D/VEGFR-3 signaling pathways may be potential therapies for primary tumors and metastasis
via the lymphatics. VEGF-A and VEGF-B, and their cognate receptor tyrosine w-kinase VEGFR-1 and VEGFR-2 are regarded as most important regulators of angiogenesis and key targets of antiangiogenesis [
20,
48]. However, there is a crosstalk between angiogenesis and lymphangiogenesis in tumor progression [
41]. Nagy et al. have demonstrated that in addition to angiogenesis, VEGF-A also induces proliferation of lymphatic endothelium, resulting in the formation of greatly enlarged and poorly functioning lymphatic channels, and abnormal lymphangiogenesis; these findings raise the possibility that abnormal lymphangiogenesis may also be expected in other circumstances such as malignant tumors characterized by VEGF-A overexpression [
32]. Thus in the design of anti-lymphangiogenesis, in addition to the VEGF-C,-D/ VEGFR-3 signaling pathways, the VEGF-A or -B/VEGFR-2 signaling pathways should be considered as potential therapy targets for primary tumors and metastasis.
A growing body of evidence has indicated that traditional Chinese medicines contain anticancer ingredient. NCTD (7-oxabicyclo [2.21] heptane-2, 3-dicarboxylic anhydride) is a demethylated derivative of cantharidin with antitumor properties, which is an active ingredient of the traditional Chinese medicine Mylabris, and is a small-molecule, low-cytotoxic compound synthesized from furan and maleic anhydride
via the Diels Alder reaction [
15,
49]. It has been reported that NCTD not only effectively inhibits the proliferation and growth of a variety of human tumor cells
in vitro and
in vivo, but also is used selectively in clinic to treat hepatic, gastric, colorectal and ovarian carcinomas and leucopenia in China because of its effective anticancer activity, fewer side effects and leukocytosis [
3,
9,
12,
19,
60]. Some experiments have also showed that NCTD plays an important role in antiangiogenesis and anti-vasculogenic mimicry for some carcinomas [
4,
51,
61‐
63]. However, the antitumor lymphangiogenic role of NCTD in tumor lymphangiogenesis and lymphatic metastasis, and the related molecule mechanism are not still elucidated, and so far no similar studies have been published. Recently, we reported the inhibitory effect of NCTD on lymphatic tube formation, i.e. lymphangiogenesis of human LECs and the underlying mechanisms
in vitro [
23]. Here, we further investigated the effects of NCTD on lymphatic tube formation of the co-culture system consisting of HCAC HT-29 cells and LECs i.e. primary human dermal lymphatic endothelial cells (HDLECs)
in vitro, tumor growth and lymphangiogenesis of the
in-situ colonic xenografts in nude mice
in vivo, and the related signaling pathways such as VEGF-C, −D/VEGFR-3 and possible crosstalk pathway VEGF-A/VEGFR-2
in vitro and
in vivo, so as to explore that it is whether served as a target inhibitor for tumor lymphangiogenesis and lymphatic metastasis, and a potential small-molecule synthetic antilymphangiogenic agent for HCACs.
Methods
Cell lines and cultures
Human colonic adenocarcinoma HT-29 cell lines were provided by the Institute of Cell and Biochemistry, Chinese Academy of Sciences (Shanghai, China), and grown in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS; Gibco, USA) in an incubator (Forma Scientific, USA) at 37 °C under a mixture of 95 % air and 5 % CO2.
Human lymphatic endothelial cells were primary HDLECs purchased from ScienCell Research Laboratories, USA. Cells were identified by immunefluorescent cytochemical technique
via CD31, Podoplanin and LYVE-1, and grown in endothelial cell growth medium (ECGM) with endothelial cell growth factor (ScienCell Research Laboratories) in an incubator (Forma Scientific) with 5 % CO
2 at 37 °C as described previously [
23], then were used in the experiments at fifth generation of the cells.
In-situ colonic xenograft assay and survival analysis in vivo
This study was carried out in strict accordance with the official of Chinese Guide for the Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting of
In Vivo Experiments) guideline [
18] in order to investigate the inhibitory effect of NCTD on HCACs by
in-situ xenograft assay and survival analysis
in vivo. The protocol was approved by the Ethics Committee of Animal Experiments of Tongji Hospital, Tongji University School of Medicine and the Science and Technology Commission of Shanghai Municipality (Permit Number: SYXK 2012–0031).
Balb/c nu/nu mice (male mice, 5 ~ 6-week old, about 20 g) from the Shanghai Laboratory Animal Center, China) were housed in specific pathogen free (SPF) condition.
In-situ colonic xenograft and the xenograft lymphangiogenic model of HT-29 cell lines in nude mice were established as described previously [
47]. Xenograft mice were randomly divided into a control group, receiving intraperitoneal (i.p.) injections of 0.2 ml sterile saline and administration through gastric tube of 0.1 ml sterile saline once two days for 6 weeks, a NCTD group, a Sorafenib group and a NCTD + Sorafenib group (20 mice per group), in which each mouse respectively received i.p. injection of NCTD (28 mg/kg, a dose of 1/5 LD
50 [
61]; No. GYZZ- H20064531, Injection solution, 5 mg/ml, Jiangsu Yew Pharmaceutical Co., Ltd, Wuxi, China) given in 0.2 ml sterile saline and administration through gastric tube of 0.1 ml sterile saline, i.p. injection of 0.2 ml sterile saline and administration through gastric tube of Sorafenib (40 mg/kg; Sorafenib Tosylate Tablets, 0.2 g/tablet, Bayer HealthCare AG, Germany) given in 0.1 ml sterile saline, or simultaneously i.p. injections of 28 mg/kg NCTD and administration through gastric tube of 40 mg/kg Sorafenib, once two days for 6 weeks in all. The xenograft size was measured with calipers two times each week. Of xenograft mice in each group, one half were sacrificed under anesthesia at 8 weeks after agent administration, tumor growth including tumor volume, tumor growth curve and tumor inhibitory rate were evaluated, and tumor morphology such as hematoxylin and eosin (H&E) staining, immunohistochemical staining and microstructures were observed under an inverted light microscope (Olympus IX70, Japan) and a TEM (JEM-1230, JEOL, Japan), respectively, as described previously [
23,
47,
61]; other half of xenograft mice continued to be housed in SPF condition, and their survivals were evaluated. Mice outcome was followed from the date of drug administration to the date of death. The median follow-up period for mice was 16 (range, 3–30) weeks.
Lymphangiogenic and lymphatic micrometastic assays of the in-situ colonic xenografts in vivo
In the experiment, tumor lymphangiogenesis and lymphatic micrometastasis of the
in-situ colonic xenografts
in vivo including lymphatic specific marker LYVE-1, D2-40 and lymphatic micrometastic marker CK20 at protein and mRNA levels, and LMVD were determined by using SABC immunohistochemical staining, western blotting and fluorescent quantitative RT-PCR as described previously [
47]. As shown in Table
1, PCR amplifications were performed with LYVE-1, D2-40 gene-specific primers designed and synthesized by Invitrogen (USA).
Table 1
Lymphangiogenic signaling-related and lymphatic specific markers
HCACCs and the co-culture system in vitro | VEGF-A | 5′-CAC CGC CTC GGC TTG TCA CAT-3′ |
5′-CTG CTG TCT TGG GTG CAT CTG-3′ |
VEGF-C | 5′-ACC TGC CCC ACC AAT TAC A-3′ |
5′-GCC TCT TGT AAA GAC TGG TT-3′ |
VEGF-D | 5′-GCT GTT GCA ATG AAG AGA CG-3′ |
5′-TCT TCT GTT CCA GCA AGT GC-3′ |
VEGFR-2 | 5′-CAC ACA GAG ATG ATT ACT ACA CTG-3′ |
5′-CCA TCT TGA GCA TCA GAT CC TC-3′ |
VEGFR-3 | 5′-AAG TAC ATC AAG GCA CGC ATC GAG-3′ |
5′-GGC TTG TTG ATG AAT GGC TGC TCA-3 |
GAPDH | 5′-ACA GAG CCT CGC CTT TGC C-3′ |
5′-CAT GTC GTC CCA GTT GGT G-3′ |
In-situ xenograft cells in vivo | VEGF-A | 5′-CTG CTC GCC GCT GCG CTG-3′ |
5′-GTG CTG GTG TTC ATG CAC TGC AG-3′ |
VEGF-C | 5′-GCC ACG GCT TATG CAA GCA AAG AT-3′ |
5′-AGT TGA GGT TGG CCT GTT CTC TGT-3′ |
VEGF-D | 5′-CGA TGT GGT GGC TGT TGC AAT GAA-3′ |
5′-GCT GTT GGC AAG CAC TTA CAA CCT-3′ |
VEGFR-2 | 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′ |
5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′ |
VEGFR-3 | 5′-GAC AGC TAC AAG TAC GAG CAT CTG-3′ |
5′-CGT TCT TGC AGT CGA GCA GAA-3′ |
CK-20 | 5′-CAG ACA CAC GGT GAA CTAT GG-3′ |
5′-GAT CAG CTT CCA CTG TTA GAC G-3′ |
LYVE-1 | 5′-TGC AGA ATT ATG GGG ATC AC-3′ |
5′-GGC TGT TTC AAC TTG GTC CT-3′ |
D2-40 | 5′-GGT GCC GAA GAT GAT GTG-3′ |
5′-CGA TGC GAA TGC CTG TTA-3 |
GAPDH | 5′-GCA CCA CCA ACT GCT TA-3′ |
5′-AGT AGA GGC AGG GAT GAT-3′ |
In the experiment, the lymphatic capillary-like structures formed from the 3-D culture of HDLECs and the co-culture system, and the expression of LYVE-1 and D2-40 from these cultures and co-cultures
in vitro were observed and determined. 24-well plates by using Transwell chambers with polycarbonate filters (pore size 0.4 μm, diameter 6.5 mm) were used. HT-29 cells (1 × 10
5 cells/ml) were added to or not added to the upper compartment of the chamber; HDLECs (5 × 10
4 cells/ml) were added to the lower compartment of the chamber in which bottom prior to the laying of Matrigel matrix (Becton Dickinson, USA) (200 μl/per chamber). The medium was changed every 2 days. After 1 week, cells were untreated (control group, equal ECGM solution) or treated with 2.5 μg/ml NCTD (NCTD group; about 1/3 IC
50 for HDLECs [
37]), 100 μl mF4-31C1 (Epitomics, USA; mF4-31C1 group) and NCTD + mF4-31C1 (NCTD + mF4-31C1 group) (6 chambers per group), respectively, in fresh culture medium in an incubator (Forma Scientific) with 5 % CO
2 at 37 °C for 2 ~ 4 days. The effects on lymphatic tube formation including the capillary-like structures, the total number of cell clusters and branching of tube formation (i.e., capillary-tube number) of each group were observed using an inverted phase-contrast light microscope (Olympus IX70) as described previously [
23]. At the same time, the expression of LYVE-1 and D2-40 from the 3-D culture or co-culture was determined using western blotting as described previously [
23,
47]. These experiments were performed in triplicate.
Proliferation and proliferating marker Ki-67 assays in vitro
Methyltiazolyl tetrazolium (MTT; Sigma, USA)-based colorimetric assay was used to evaluate the inhibitory effect of NCTD on proliferation of HT-29 cells, HDLECs and the co-culture system in vitro. The cultures were divided into a NCTD group and a control group. HT-29 cells (1 × 105 cells/ml, 100 μl/well) were cultured in 24-well plates in RPMI-1640 medium (100 μl/well), and HDLECs (5 × 104 cells/ml, 100 μl/well) were cultured in fibronectin-coated 24-well plates in ECGM medium (100 μl/well). Proliferation assay for the co-culture system, 24-well plates by using Transwell chambers with polycarbonate filters (pore size 0.4 μm, diameter 6.5 mm) were used; HT-29 cells (1 × 105 cells/ml) were added to the upper compartment of the chamber, HDLECs (5 × 104 cells/ml) to the lower compartment of the chamber (200 μl/per chamber). Cells then were untreated (control group, equal RPMI-1640 or ECGM solution) or treated with various concentrations (1.25 ~ 100 μg/ml; 6 wells per concentration) of NCTD (NCTD group) in fresh culture medium at 37 °C in 5 % CO2 for 24 h. The optical densities (A value) at 490 nm were measured with an enzyme-linked immunosorbent assay reader (Elx800UV, Bio-Tek, USA). The A490 value of the experimental groups was divided by the A490 value of untreated controls and presented as a percentage of the cells. The inhibitory percent of NCTD on the cells (%) = (1- A490 value in the experimental group/A490 value of control group) × 100 %. Three separate experiments were performed. The concentration of drug giving 50 % growth inhibition (IC50) was calculated from the formula IC50 = lg−1{Xm-I [P-(3-Pm-Pn)/4]}.
In order to further observe the inhibitory effect of NCTD on proliferation of HDLECs and the co-culture system, proliferation marker Ki-67 of above LYVE-1 or D2-40-positive HDLECs and co-culture system in
vitro were determined by SABC immunocytochemical staining as described previously [
4]. Cells plated on slides were untreated (control group, equal RPMI-1640 or ECGM solution) or treated with an 1/3 IC
50 dose of NCTD (NCTD group), and primary antibody of Ki-67 (mouse monoclonal antibody, 1:100, Antibody Diagnostica Co., USA) was added, then biotinylated secondary antibody (goat anti-rabbit IgG, 1:100), SABC reagents and DAB solution (all from Boster Co., China). For negative control, the slides were treated with PBS in place of primary antibody. Then, cells were rinsed in distilled water, dehydrated through alcohol and xylene and mounted on a coverslip using a permanent mount medium for analysis by a microspectrophotometer (Leitz Dmrbe, Leica). Ten sample slides in each group were chosen for analysis. More than 10 visual fields were observed or more than 500 cells were counted per slide. The positive index of Ki-67 represented expression of Ki-67 protein. The stain integral of Ki-67 protein was counted according to the positive number and the intensity of staining of the cells.
Apoptosis and apoptotic gene Bcl-2 assays in vitro
Immunofluorescent dye, FCM and TEM were used in this assay as described previously [
23]. Cell culture and experiment were performed according to above proliferation assay. For immunofluorescent dye, cells were fixed, washed and stained with 0.5 ml fluorescence agent Hoechst 33258 (Sigma) and CY3 NHS ester (Lumiprobe, USA), then observed and counted for cell apoptotic percent of each group under a fluorescence microscope (Nikon Eclipse TE2000-U, Japan) as described previously [
37]. For FCM, cells (5 × 10
5 cells/ml) suspended in 500 μl binding buffer were used for DNA stain with 5 μl Annexin V-FITL and propidium iodine (PI, Sigma); then, DNA value, cell cycle and apoptotic rate of each group were determined by a cell apoptotic detection kit (BioDev, China) and a fluorescent activated cell sorter (420 type FCM, Becton-Dickinson, USA) as described previously [
9,
12,
23]. Cells were observed under an inverted microscope (Olympus IX70) and a TEM (JEM-1230, JEOL) as described previously [
23].
In addition, in order to further observe the inducing effect of NCTD on apoptosis of HDLECs and the co-culture system, anti-apoptotic gene Bcl-2 of HDLECs and co-culture system in
vitro were determined by SABC as described previously [
9]. Cells plated on slides were untreated (control group, equal RPMI-1640 or ECGM solution) or treated with an 1/3 IC
50 dose of NCTD (NCTD group), and primary antibody of Bcl-2 (rabbit polyclonal antibody, 1:50, Santa Cruz, USA), biotinylated secondary antibody (goat anti-rabbit IgG, 1:100), SABC reagents and DAB solution (all from Boster Co., China) were in turn added. Then, slides were rinsed, dehydrated, mounted and observed under a microspectrophotometer (Leitz Dmrbe). For negative control, the slides were treated with PBS in place of primary antibody. Ten sample slides in each group were chosen for analysis. The positive index of Bcl-2 represented expression of Bcl-2 protein.
Migration assay in vitro
Transwell migration chambers i.e., 24-well plates by Transwell chambers with polycarbonate filters (pore size 8 μm, diameter 6.5 mm) were used in this assay. HT-29 cells (1 × 105 cells/ml) or HDLECs (5 × 104 cells/ml) were inoculated in the upper compartment of the chamber (200 μl/chamber), 0.8 ml RPMI-1640 medium with 10 % FBS or ECGM medium was added to the lower compartment of the chamber (200 μl/chamber). For the co-culture system, HDLECs were added to the upper compartment of the chamber (200 μl/per chamber), HT-29 cells to the lower compartment of the chamber (200 μl/per chamber) in 0.8 ml of RPMI-1640 medium with 10 % FBS. Cells were untreated (control group, equal ECGM solution) or treated with above 1/3 IC50 NCTD (NCTD group; 18.7 μg/ml for HT-29 cells, 2.5 μg/ml for HDLECs, 5.3 μg/ml for co-culture), 100 μl mF4-31C1 (mF4-31C1 group) and NCTD+ mF4-31C1 (NCTD + mF4-31C1 group) (6 chambers/per group), respectively, in fresh culture medium (chambers/per group) at 37 °C in 5 % CO2 for 24 h. Total number of migrating cells were measured and counted in five independent microscopic visual fields (×100) under an inverted microscope (Nikon TS100, Japan), and expressed as mean number per one field. Each experiment was performed thrice.
Invasion assay in vitro
Matrigel invasion chamber i.e. 24-well plates by Transwell chambers with polycarbonate filters (pore size 8 μm, diameter 6.5 mm) coated on the upper side with Matrigel (Becton Dickinson were used in this assay. HT-29 cell (1 × 105 cells/ml) or HDLEC (5 × 104 cells/ml) suspensions were transferred to the upper compartment of the chamber (200 μl/every chamber), while 0.8 ml RPMI-1640 medium with 10 % FBS or ECGM medium was added to the lower compartment of the chamber. For the co-culture system, HDLECs were added to the upper compartment of the chamber, HT-29 cells to the lower compartment of the chamber (200 μl/every chamber) in 0.8 ml of RPMI-1640 medium with 10 % FBS. Cell experiment was performed as above migration assay. The number of invading cells through the filter was counted after H&E staining and plotted as the mean number of invading cells per optic field in three independent experiments.
Determination of VEGF-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3 in vitro and in vivo
The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 at protein and mRNA levels from the 3-D culture of HDLECs or the co-culture system
in vitro, and the
in-situ xenografts
in vivo were determined by S-P staining, western blotting and fluorescent quantitative RT-PCR as described previously [
23,
47].
For S-P staining, slides were treated according to the kit brochure (Jinmei Biotechnology Co., Ltd., Shanghai), added in order with primary antibody [rabbit anti-human monoclonal antibody VEGF-A (Santa Gruz), VEGF-C (Invitrogen), VEGF-D (Abcam, USA), VEGFR-2 (Cell Signaling, USA), VEGFR-3 (Cell Signaling), biotinylated anti-rabbit secondary, HRP logo Streptavidin and DAB solution, respectively. Then, slides were rinsed, dehydrated, mounted and observed under an optic microscope (Olympus, Japan). For negative control, the slides were treated with PBS in place of primary antibody. Six sample slides in each group were chosen by analysis. Visual fields (>10) were observed or >500 cells were counted per slide.
Lowry method protein kit (Puli Lai Co., Shanghai) were used for western blotting according to the kit brochure. An aliquot of 20 mg of proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. One hour after being blocked with PBS containing 5 % non-fat milk, the membrane was incubated overnight, was then added in order with each primary antibody [anti-VEGF-A, anti-VEGF-C, anti-VEGF-D (Abcam), and anti-VEGFR-2, anti-VEGFR-3, anti-β-actin (Cell Signaling)], HRP-labeled secondary antibody (Abcam) (all 1:1000)], HistoFine (Dako, Glostrup, Denmark) for 2 h. The target proteins were visualized by an enhanced chemiluminescent reagent (GE Healthcare, USA), imaged on the Bio-Rad chemiluminescence imager. The gray value and gray coefficient ratio of each protein was analyzed and calculated.
Fluorescent quantitative RT-PCR was performed as described by the manufacturer. Total RNA was extracted using the TRIzol reagent (Invitrogen). The primers for amplification were designed and synthesized by Sangon Co., Shanghai. The primers for VEGF-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3 and GAPDH
in vitro and
in vivo were as shown in Table
1. RT-PCR reaction conditions and the amplifying conditions
in vitro were as described previously [
23]. GAPDH was used as an internal control, with the annealing temperature of 56 °C for 40 cycles (94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 10 min). PCR products (10 μl) were placed onto 15 g/L agarose gel and observed by ethidium bromide staining using the ABI PRISM 7300 SDS software. The relative mRNA expression levels was calculated by the formula (relative mRNA expression = 2
-△△Ct).
Statistical analysis
Statistical analyses were performed using SPSS 13.0 and Microsoft Excel Office 2007 for Windows. All data were presented as mean ± SD. Statistical differences were evaluated using Student’s t test or the Chi-square test. P < 0.05 was considered statistically significant. Survival curves were calculated with the Kaplan-Meier method and were compared using the log-rank test.
Discussion
As a small-molecule synthetic compound, NCTD has been reported to possess multiple potent antitumor properties in several cell lines, tumor xenograft models and human carcinomas; and is used selectively in clinic to treat many human malignant tumors by oral, intravenous injection or drip, or as a premedication or in combination with chemotherapy drugs for improving efficacy and reducing side effects in China because of its anticancer activities, fewer side effects and leukocytosis [
3,
4,
9,
12,
15,
19,
23,
38,
51,
60‐
63]. However, these studies merely focused on effects of NCTD on the proliferation, apoptosis, growth and angiogenesis of a variety of human tumor cells
in vitro and
in vivo, including hepatoma HepG2 cells [
55,
58], gallbladder cancer GBC-SD cells [
8,
9,
12,
51,
61,
63], colon cancer HT29 cells [
34], leukemia K562 [
59] and HL-60 cells [
21], and melanoma A375-S2 [
1], and so on. In the experiment, on the base of our recent report in which NCTD suppressed lymphangiogenesis in human LECs [
23], we further investigate the effect of NCTD on tumor lymphangiogenesis, and present evidence that NCTD inhibits tumor growth and lymphangiogenesis of HCACs by downregulating VEGF-C,-D/VEGFR-3 and VEGF-A/VEGFR-2 signaling pathways. This result is supported by observations listed below: 1) lymphatic tube formation from the 3-D culture of HDLECs and the co-culture system is observed
in vitro; NCTD inhibits this lymphatic tube formation by suppressing proliferation, migration, invasion, tube formation of HDLECs in the co-culture system, inhibiting the expression of proliferation marker Ki-67 and anti-apoptotic gene Bcl-2, induced S-phase cell cycle arrest and cell apoptosis; 2) tumor lymphangiogenesis and lymphatic micrometastasis exist in the
in-situ colonic xenografts
in vivo; NCTD inhibits these tumor lymphangiogennesis and lymphatic metastasis; 3) VEGF-C, VEGF-D and VEGFR-3, and VEGF-A, VEGFR-2 proteins/mRNAs are highly expressed in the co-culture system
in vitro and the
in-situ colonic xenografts
in vivo; whereas NCTD inhibits tumor growth and lymphangiogenesis by downregulating the expression of VEGF-C, VEGF-D and VEGFR-3, and VEGF-A, VEGFR-2 proteins/mRNAs. These results strongly support that NCTD serves as a potential antilymphangiogenic agent for tumor lymphangiogenesis of HCACs.
As the growth of newly lymphatic vessels in cancer, tumor lymphangiogenesis acts as a conduit by which disseminating tumor cells access regional lymph nodes, and lymph node tumor lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes actively promote metastasis
via the lymphatics [
25,
31,
43]. It was reported that tumor lymphangiogenesis and the presence of metastatic cells in the sentinel lymph node are prognostic indicators of metastasis to lymph nodes, and the degree of dissemination determines the therapeutic course of action [
64]. Thus, inhibition of tumor-associated lymphatic and lymphangiogenesis may be a key for controlling of primary tumor and metastasis [
20,
25,
28,
31,
39,
43]. So, recent researchers have focused on hot-spots of antitumor by inhibiting tumor lymphangiogenesis [
20,
28,
37,
64]. Some tumor lymphangiogenic inhibitors such as deguelin, endostar, silencing Id-1, liposomal honokiol, mF4-31C1 and sVEGFR3-Ig have been reported as adjuvant antilymphangiogenic and antitumor drugs against some metastatic cancers in experiment and in clinic setting [
6,
7,
14,
16,
26,
35,
52]. But, there is still no experimental and clinical evidence whether NCTD can be used as a potential tumor lymphangiogenesis inhibitor. In this experiment, we further confirmed that NCTD not only has the antilymphangiogenic effect on human LECs, but also has the antilymphangiogenic activity against tumor growth and tumor lymphangiogenesis of HCACs
via the 3-D culture of HDLECs and the co-culture system
in vitro, and the
in-situ colonic xenografts in nude mice
in vivo.
Lymphatic tube formation i.e. lymphangiogenesis is a critical step for tumor lymphangiogenesis; whereas early stage of this lymphangiogenesis includes cell proliferation, apoptosis, migration and invasion of LECs [
23,
25,
31,
43]. As a useful specific marker for detecting lymphangiogenesis or lymphatic micrometastasis and circulating carcinoma cells, LYVE-1 expressed on the surface of LECs plays an important role in the transportation of both lymphocytes and tumor cells into the lymphatic system, is considered to be the most valuable marker in identifying LECs and tissue lymphatics [
17,
36]; Like LYVE-1, D2-40 (Podoplanin), which is an integrated cytoplasm/cell membrane mucoprotein mainly expressed in micro-LECs and lymphatic vessels and a relatively high specificity marker for LECs and lymphatic vessels, has now been used as value tools for the detection or prediction of tumor lymphangiogenesis and lymph node metastasis in human carcinomas [
2,
13]; CK20, a type I cytoskeletal protein located on chromosome 17q21.2, have been known as a useful marker for detection of lymphatic micrometastasis and circulating carcinoma cells or for discrimination of the origin of metastatic tumors of unknown primary location such as micrometastasis in regional or sentinel lymph nodes and blood [
29,
53,
54]. LMVD is the lymphatic microvessel density. Quantification of LMVD in the tumor might also be important for the evaluation of lymphangiogennesis, occult nodal metastasis (micrometastasis) and lymphatic metastasis in human carcinomas [
40,
50]. We previously detected LYVE-1 expression, lymphatic microvessel and LMVD in human colorectal cancers, and evaluated their correlation with lymphangiogennesis, lymphatic metastasis, VEGF-C, −D and VEGFR-3, and prognosis in patients with colorectal cancer [
10,
11,
22]. As a soluble VEGFR-3 antibody, mF4-31C1 has been shown the antilymphangiogenic activity for human LECs
in vitro and the anti-tumor lymphangiogennesis
in vivo [
23,
36]. Sorafenib (Nexavar, a novel bi-aryl urea BAY 43–9006), is an oral multikinase inhibitor that blocks tumor cell proliferation and carcinogenesis by targeting the Raf/MEK/ERK signaling pathway and exerts an antiangiogenic effect by targeting several receptor tyrosine kinases including VEGFR-2, VEGFR-3 and platelet-derived growth factor receptor (PDGFR)-beta [
37,
38]. Sorafenib has been demonstrated potent antitumor activities in studies
in vitro, preclinical xenograft models of different tumor cells including human colonic adenocarcinoma cells
in vivo and human clinical trials, has been clinically used for patients with advanced hepatocellular carcinoma, non-small-cell lung cancer, renal cancer and colonic cancer and demonstrated an improved overall survival in the patients [
5,
44,
45]. Furthermore, Sorafenib has been used to enhance the antitumor effects and to overcome resistance by combination with chemoembolisation, chemoradiation and other antiangiogenic agents with different action mechanisms [
30,
42,
56]. In view of the antilymphangiogenic activity of mF4-31C1 and the antitumor angiogenic or lymphangiogenic effects (targeting several receptor tyrosine kinases, including VEGFR-3) of Sorafenib, mF4-31C1 and Sorafenib were chosen as the experimental controls in this study. The results showed that NCTD, not only as mF4-31C1
in vitro [
14,
23,
26,
35], inhibited the lymphatic tube formation of the 3-D culture of HDLECs and the co-culture system by suppressing proliferation, migration, invasion, and inducing apoptosis, as Sorafenib
in vivo [
37,
38,
62], inhibited tumor growth and lymphangiogennesis of
in-situ colonic xenografts with a prolonged survival time (log-rank test,
P = 0.026), but also downregulated the expression of LYVE-1, D2-40 and CK20 proteins/mRNAs, decreased the LMVD numbers in the
in-situ colonic xenografts. Therefore, we believe that NCTD inhibits tumor growth and lymphangiogennesis of HCACs.
Tumors promote lymphangiogenesis by multifactor and complicated molecular mechanisms i.e., secreting molecules or cytokines such as lymphangiogenic growth factors from tumor and/or stromal cells that stimulate LEC growth and tumor lymphangiogennesis [
20,
28,
37]. These lymphangiogenic growth factors and the regulators contributing to lymphangiogenesis mainly include VEGF-C, VEGF-D and VEGFR-3 [
20,
25,
28,
37,
39]. Recently, VEGF-C, VEGF-D and their cognate receptor VEGFR-3 located on LECs and the VEGF-C, −D/VEGFR-3 signaling pathway are believed to be the most important lymphangiogenic growth factors and the most pivotal mechanism underlying tumor lymphangiogennesis [
20,
25,
28,
37,
39]. VEGF-C or VEGF-D combines with VEGFR-3 located on LECs, thereby promoting proliferation of new lymphatic capillaries and tumor lymphangiogenesis
via a series of intracellular signaling pathways. Clinical trials have indicated that the expression of VEGF-C, VEGF-D or VEGFR-3 is associated with lymph node metastasis and the poor prognosis of patients with cancer [
10,
11,
22]. Relevant animal models have confirmed that VEGF-C, VEGF-D or VEGFR-3 plays an important role in the regulation of tumor lymphangiogenesis and lymph node metastasis. Activation of VEGF-C, −D/VEGFR-3 axis increases motility and invasiveness of LECs, promote formation of tumor lymphangiogenesis [
20,
28,
37,
46]. So, some targeted strategies to block tumor lymphangiogenic pathways such as VEGF-C, −D/VEGFR-3 signaling pathway seem to be attractive anticancer treatment strategys. It was recently reported that Silencing Id-1 inhibits lymphangiogenesis through downregulation of VEGF-C in oral squamous cell carcinoma [
6]; that liposomal honokiol and deguelin suppresses tumor lymphangiogenesis and lymphatic metastasis in xenograft tumor models by downregulation of VEGF-D both
in vitro and
in vivo [
16,
52]; that blocking the expression of VEGFR-3 using interference vector-based RNA interference inhibits tumor growth of colorectal cancer [
26]; that mF4-31C1 specifically inhibited both physiologically normal and tumor VEGF-C-enhanced lymphangiogenesis and new lymphatic growth in a 3-D culture of LECs and a mouse model of lymphatic regeneration by targeting VEGFR-3 [
35]. Sorafenib blocked tumor proliferation and carcinogenesis by targeting inhibition of multi-signal pathways including VEGFR-3 [
37,
38]. We lately reported NCTD, as mF4-31C1, inhibited the lymphangiogenesis of human LECs by simultaneously blocking VEGF-C, −D/VEGFR-3 pathways [
23]. Actually, other than VEGF-C, VEGF-D and VEGFR-3, many other potent growth factors were involved in tumor lymphangiogenesis, such as VEGF-A, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), angiopoietin (Ang)-1, Ang-2, insulin-like growth factor (IGF)-1 and basic fibroblast growth factor (bFGF), cyclooxygenase-2 (COX-2), and Slit2 [
20,
24,
28,
32,
33,
37,
41,
46,
48,
57]. It is reported that Slit2, as a potent lymphangiogenic factor, contributes to tumor lymphatic metastasis [
57]; VEGF-A treated lymphatic endothelial cell exhibited STAT3 activation in the nucleus, thereby enhancing lymphatic endothelial cell migration and increased tube formation (63). Indeed, many other growth factors such as VEGF-A, FGF-2, Ang-1, IGF-1 and HGF stimulate lymphangiogenesis indirectly through VEGF-C, VEGF-D or VEGFR-3 [
20,
27,
28,
32,
33,
41,
48]. In the present experiments, we detected the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3
via immunohistochemistry staining, western blotting and RT-PCR. The results have shown that NCTD not only downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 proteins/mRNAs but also inhibited the expression of VEGF-A and VEGFR-2 proteins/mRNAs of the co-culture system
in vitro and the
in-situ colonic xenografts
in vivo; the downregulation of VEGF-C, −D/VEGFR-3 and VEGF-A/VEGFR-2 by NCTD in combination with mF4-31C1/Sorafenib can be enhanced. Thus, we believe that NCTD inhibit tumor growth and lymphangiogenesis of the co-culture system
in vitro and the
in-situ colonic xenografts
in vivo by simultaneously blocking VEGF-C, VEGF-D and VEGFR-3 other than VEGF-A and VEGFR-2. Because there is a crosstalk between angiogenesis and lymphangiogenesis in tumor progression, we thus deduce that NCTD inhibits tumor growth and lymphangiogenesis of HCACs, as mF4-31C1 or Sorafenib through directly downregulating the VEGF-C, −D/VEGFR-3 signaling pathway, or similar to Sorafenib through indirectly downregulating the VEGF-A/VEGFR-2 signaling pathway. The present findings may be of importance to explore the therapeutically strategy of NCTD as an antilymphangiogenic agent for tumor lymphangiogenesis and lymphatic metastasis.
Authors’ contributions
LXP, JW, SJJ, and FYZ designed the research, analyzed the data and wrote the manuscript. JW and SJJ carried out in vitro experiments and 3-D culture of the cells. LXP, JW and SJJ carried out in vivo experiments of the xenografts. LZY, ZJT, SW, and ZW were responsible for the detection of the related markers, and the observation of microscope and TEM, respectively. FYZ is the guarantor. All authors have read and approved the final manuscript.